The objective of this project is to evaluate the safety and efficacy of drug resistance gene therapy utilizing methylguanine methyltransferase (MGMT) combining in vitro and murine studies with a clinically relevant large animal model to develop safer gene therapy vectors for clinical applications. We have demonstrated efficient in vivo selection achieving high gene marking levels (>90%) and chemo-protection of the hematopoietic system in the canine model after delivering MGMT to repopulating cells using gammaretroviral and lentiviral vectors (see preliminary data). This suggests that MGMT-mediated in vivo selection will benefit a broad range of genetic diseases and also elicit a significant therapeutic benefit of bone marrow chemo-protection in patients with malignant disease. In our initial analysis, gammaretroviral integrants showed the most significant increase very close to transcription start sites and a higher frequency in and near proto-oncogenes relative to lentiviral integrants, suggesting that gammaretroviral vectors may be the most prone to adverse gene activation. Thus it will also be important to develop strategies to reduce the potential of transformed clones and to have a platform in place to test ongoing developments in vector design aimed at safety. Therefore, to develop safer gene therapy vectors we will conduct a comprehensive analysis of the provirus integration profile of canine cells gene-modified with lentiviral MGMT vectors containing strong viral promoter/enhancers before and after in vivo selection as an initial assessment of current lentivirus vector safety. We will determine if selection increases the percentage of retroviral integration sites near proto-oncogenes. As a strategy to reduce the risk of promoter/enhancer activation, we will characterize and test chemotherapy-inducible promoter systems and incorporate the most promising constructs into lentivirus self-inactivating vectors (SIN) to examine their genotoxicity in the murine model before testing optimum vectors in the canine model. This proposal will combine in vitro and murine studies to develop and characterize safer gene therapy vectors and utilize the canine model to evaluate the efficacy and safety of MGMT-mediated selection in a clinically relevant large animal model.